GSM7977648: Central forebrain, ZT18 undesturbed, rep1 [ZT18-1]; Rattus norvegicus; RNA-Seq
Rats were decapitated by guillotine under normal room light (ZT2–10) or under dim red light (ZT14–22). Following decapitation, forebrains were resected (Fig. 1A), frozen in 2-methylbutane suspended in dry ice, and then stored at -80 °C until homogenization for RNA extraction. Just before RNA isolation, forebrains were removed from -80 °C storage and placed on dry ice. Prior to use, a stainless-steel mortar and pestle were cleaned with RNase Zap (Thermo Fisher) and 70% ethanol. The mortar was then partially filled with liquid nitrogen before a forebrain was added, pulverized, and placed in a conical tube. Between each sample, the mortar and pestle were cleaned with 70% ethanol. A small aliquot of sample was removed for RNA isolation using Trizol Reagent (Invitrogen), according to the manufacturer's instructions. Purified RNA was resuspended in water, and concentration and purity were measured with a Nanodrop spectrophotometer (Thermo Fisher). WTTS-Seq libraries were prepared as described by Zhou et al. https://doi.org/10.1534/genetics.116.188508. Briefly, total RNA (2.5 µg) was incubated at 70 °C with 10X Fragmentation buffer (Invitrogen) for 3 min. The fragmentation reaction was halted by the addition of Stop Solution and incubation on ice for at least 2 min. Next, poly(A)+ RNA was purified from the fragmented total RNA with Dynabeads Oligo (dT)25 (Invitrogen), according to the manufacturer's directions, and used for first-strand cDNA synthesis in a 20 µL reaction mixture. First, 1.0 µL of barcode primer (100 µM) and 1.0 µL of a common SMART primer (100 µM) were annealed to the poly(A)+ RNA template by heating to 65 °C for 5 min and incubating on ice for at least 2 min. Next, 4.0 µL of 5X First-strand buffer (Invitrogen), 1.0 µL of SuperScript III reverse transcriptase (Invitrogen), 1.0 µL of 0.1 M dithiothreitol, 2.5 µL of 10 mM dNTP, and 1.0 µL of RNase OUT (Invitrogen) were added to the mixture. First-strand cDNA was synthesized by incubating the mixture at 40 °C for 90 min in the presence of library-specific adaptors. Synthesis was terminated by heating the mixture at 70 °C for 15 min. RNases I (100 U/µL; Invitrogen) and H (2 U/µL; Invitrogen) were subsequently added and incubated with the mixture at 37 °C for 30 min to hydrolyze the remaining single-stranded RNA molecules and ensure that only single-stranded cDNA remained. RNase activities were terminated by heating the samples at 70 °C for 20 min. Following purification with solid-phase reversible immobilization (SPRI) beads, second-strand cDNA was synthesized from first-strand cDNA by asymmetric PCR. In addition to the cDNA, the 50 µL PCR reaction contained 1.0 µL of Phusion Hi-Fidelity DNA polymerase, 10.0 µL of 5X HF buffer, 1.0 µL of 0.4 µM barcode primer, 1.0 µL of 0.8 µM common primer, 1.0 µL of 10 mM dNTP, and nuclease-free water. The PCR reaction was carried out by heating at 95 °C for 30 sec, followed by 20 cycles of 98 °C for 10 s, 50 °C for 30 s, and 72 °C for 30 s, with a final elongation step at 72 °C for 10 min. SPRI beads were used to purify and select 200 – 500 bp fragments from the final library.